Elevated GRO-α and IL-18 in serum and brain implicate the NLRP3 inflammasome in frontotemporal dementia

Neuroinflammation is a hallmark of frontotemporal dementia (FTD), a heterogeneous group of proteinopathies characterized by the progressive degeneration of the frontal and temporal lobes. It is marked by microglial activation and subsequent cytokine release. Although cytokine levels in FTD brain and CSF have been examined, the number of cytokines measured in each study is limited and knowledge on cytokine concentrations in FTD serum is scarce. Here, we assessed 48 cytokines in FTD serum and brain. The aim was to determine common cytokine dysregulation pathways in serum and brain in FTD. Blood samples and brain tissue samples from the superior frontal cortex (SFC) were collected from individuals diagnosed with behavioral variant FTD (bvFTD) and healthy controls, and 48 cytokines were measured using a multiplex immunological assay. The data were evaluated by principal component factor analysis to determine the contribution from different components of the variance in the cohort. Levels of a number of cytokines were altered in serum and SFC in bvFTD compared to controls, with increases in GRO-α and IL-18 in both serum and SFC. These changes could be associated with NLRP3 inflammasome activation or the NFκB pathway, which activates NLRP3. The results suggest the possible importance of the NLRP3 inflammasome in FTD. An improved understanding of the role of inflammasomes in FTD could provide valuable insights into the pathogenesis, diagnosis and treatment of FTD.

Neuroinflammation is recognized as a hallmark for neurodegenerative diseases including frontotemporal dementia (FTD), a heterogeneous group of neurodegenerative clinical syndromes characterized by progressive behavioral and/or language changes and associated cognitive deficits 1 . There are three clinical subtypes of FTD: behavioral variant FTD (bvFTD), nonfluent variant primary progressive aphasia and semantic variant primary progressive aphasia, with bvFTD being the most common 2 . Neuropathologically, FTD is categorized based on the pathological cellular inclusions, with tau and Tar-DNA binding protein-43 (TDP-43) being the most prevalent, and both of which are known to trigger neuroinflammation 3,4 . Neuroinflammation is primarily mediated by activated astrocytes and microglia, the resident immune cells of the central nervous system. Microglia are mainly responsible for maintaining homeostasis and mediating host defense against pathogens and toxic protein aggregates. These stimuli trigger the activation of microglia, which releases pro-inflammatory cytokines, chemokines and reactive oxygen species (ROS). While early studies referred to four primary features of neuroinflammation-microglial activation, increased cytokines/chemokines, recruitment of peripheral immune cells and local tissue damage-the definition for this term has since broadened to cover most immune processes in the nervous system 5 . Physiologically, consequences of neuroinflammation include elevated ROS/oxidative stress, neuronal cell death, impaired phagocytosis and autophagy, mitochondrial dysfunction and protein aggregation [6][7][8][9] , all of which are known to contribute to the pathogenesis of neurodegenerative diseases [10][11][12][13] .
Neuroinflammation in FTD brain is evident by the presence of activated microglia in disease-affected regions, as shown by immunohistochemistry [14][15][16][17][18] . Indeed, the presence of activated microglia in the brain of FTD patients have been confirmed in vivo by positron emission topography (PET) using inflammation markers C-PK11195 Participant brain tissues. A different cohort of bvFTD patients and controls (Table 2) was used for the brain tissue analysis. Fresh-frozen post-mortem brain tissue samples were obtained with consent from the Sydney Brain Bank at Neuroscience Research Australia and NSW Brain Tissue Resource Centre at the University of Sydney (both brain banks ethically approved through their institutions to collect, characterize and bank brain tissue for research purposes). Ethics approval for this tissue study was from the University of New South Wales Human Research Ethics (approval number: HC15789). All brain donors underwent standardized assessments in life and standardized neuropathological examination, and met current consensus diagnostic criteria for sporadic bvFTD with TDP-43 pathology 63,64 or no significant neuropathology (controls) 65 1  Male  66  39  Early FTD-TDP   2  Male  62  15  Early FTD-TDP   3  Female 72  25  Early FTD-TDP   4  Male  61  37  Early FTD-TDP   5  Female 65  22  Late FTD-TDP   6  Female 84  17  Late FTD-TDP   7  Male  60  28  Early FTD-TDP   8  Female 99  13  Late FTD-TDP   9  Female 86  25  Late FTD-TDP   10  Male  74  20 Late FTD-TDP www.nature.com/scientificreports/ lects brain tissue from brain donors participating in the FRONTIER brain donor program approved through the South Eastern Sydney Local Health District Human Research Ethics (approval number: HREC 10/092) and so the bvFTD cases with brain tissue were clinically assessed via the same procedures as indicated for the patient blood serum. Tissue samples from the superior frontal cortex were collected from ten bvFTD cases (5 male, 5 female) 67 and 11 controls (5 male, 6 female) 68 . The mean age of the two groups were 72.9 and 79.5 years, respectively. Pathological severity of FTD was also assessed within the bvFTD group which could be split further into early stage 1 (N = 5) versus later stage disease (N = 5) 69 , reflecting the average disease durations of these two subgroups (mean ± standard deviation for stage 1 disease duration of 2.7 ± 1.5 years versus stage 2/3 disease durations of 8 ± 4 years).
Protein fraction extraction from human brain tissues. The TBS fractions, which contain the cytosolic proteins, were used in cytokine analysis. The TBS fractions were extracted from the superior frontal cortex as previously described 68  Statistical analysis. All statistical analyses were performed using SPSS statistical software (IBM, Chicago, IL, United States). A multivariate analysis (general linear model), covarying for age and sex, was used to determine differences in the cytokine levels in FTD (N = 10) and control (N = 11) with posthoc statistical significance set at P < 0.05. Principal component factor analyses (PCA) were performed to determine if significantly altered cytokines, were clustering in the same group of variance for serum and brain cytokines. To be considered significant, cytokines required a loading score of > 0.7 and to be responsible for > 10% of variance. PCA was first performed on serum cytokines to determine if cytokines that were altered in the serum of bvFTD cohort were clustering together. This is followed by analysis on brain cytokines to examine if similar components of variance were observed in both brain and serum.

Results
Altered cytokine levels in FTD serum. Forty-eight cytokines were measured in bvFTD (N = 10) serum and controls (N = 10) using a multiplex assay. Two samples, year 1 and year 2 (i.e. 12-months apart), from each individual were assessed. Firstly, we assessed the cytokines independent of time and found that IL-2Rα, IP10, macrophage inflammatory protein 1-alpha (MIP-1α) and stem cells growth factor-beta (SCGF-BB) were significantly increased in bvFTD compared to controls ( Fig. 1). Of the 48 cytokines, IL-10, IL-12 (p40), IL-5, IL-15, IL-16, monocyte chemotactic protein-3 (MCP-3) and vascular endothelial growth factor (VEGF) were not detected by the multiplex assay. Secondly, we assessed the cytokines longitudinally and found that five chemokines (GRO-α/CXCL1, monocyte chemotactic protein-1 (MCP-1), macrophage inflammation protein 1-beta (MIP-1β), RANTES and SDF-1α) ( Fig Cytokine analysis of FTD brain tissue. We were also interested in changes in the cytokines in bvFTD brain and assessed the same cytokines, using the same multiplex assay, in the superior frontal cortex, a diseaseaffected region, of FTD (N = 10) and controls (N = 11). Of the 48 cytokines, HGF and IL-18 were significantly elevated in bvFTD compared to controls ( Fig. 4) with IL-5 being undetectable. We then categorized bvFTD into two groups based on neuropathological severity, i.e. early stage 1 bvFTD (N = 5) and later stage 2/3 bvFTD (N = 5), and divided the cytokines into functional groups, i.e. chemokines, interleukins, interferons, growth factors and colony stimulating factors. In terms of chemokines and interleukins, GRO-α ( Fig. 5A) and IL-16 ( Fig. 5B) were significantly elevated in late bvFTD compared to controls, whereas IL-18 was more significantly increased in early bvFTD relative to late bvFTD (Fig. 5B). In terms of interferons, there were no changes in either early or late bvFTD compared to controls (Fig. 6A). In terms of growth factors, HGF was increased in early bvFTD and further increased in late bvFTD compared to controls (Fig. 6B). In summary, GRO-α and IL-18 are elevated in both serum and brain in bvFTD compared to controls. www.nature.com/scientificreports/

Discussion
Neuroinflammation is known to play a major role in the neuropathology of neurodegenerative diseases including bvFTD 24 . Since cytokines are integral to neuroinflammation, an improved understanding of cytokines in bvFTD could provide valuable insights into the pathogenesis of bvFTD and other neurodegenerative diseases. While changes in brain/CSF cytokines levels in bvFTD have been well documented, reports on serum cytokine concentrations in bvFTD are scarce, and the number of cytokines measured in these studies small. The present study is the first to directly compare the serum and brain levels of a comprehensive range of cytokines in bvFTD. The side-by-side assessment of serum and brain cytokine concentrations allowed the identification of common cytokines that are altered in both the blood and brain. The relative ease of serum collection over that of CSF makes these cytokines good candidates for neuroinflammation biomarkers. In addition, the grouping of functionally similar cytokines enabled the identification of novel neuroinflammation pathways that could contribute to the pathogenesis of bvFTD.
In agreement with previous studies, this study also showed cytokine level changes in serum and brain of bvFTD patients compared to those of healthy controls. In the serum, the concentrations of four cytokines, IL-2Rα, IP-10, MIP-1α and SCGF-BB, were elevated in bvFTD compared to controls, while GRO-α, IFN-α2, IL-18, MCP-1, MIP-1β and PDGF-BB have shown time-dependent increase in serum concentration with disease progression. Of note, GRO-α and IL-18 levels were also increased in bvFTD brain, in addition to HGF and IL-16. The fact that both bvFTD brain and serum have elevated levels of GRO-α and IL-18 suggests that these www.nature.com/scientificreports/ two cytokines are involved in pathways crucial in the pathogenesis of bvFTD. Principle component analysis on brain cytokines levels have placed GRO-α and IL-18 in the group 2 of brain cytokines ( Table 4), suggesting that these two cytokines are functionally related in bvFTD brain. IL-18 and GRO-α (also known as CXCL1) are both pro-inflammatory cytokines. IL-18 is involved in the activation of mast cells and CD8 + T cells, production of IFN-γ and Th2 cytokines and inducing innate-type allergic inflammation 70 while GRO-α binds to its receptor CXCR2 to promote neutrophil recruitment and activation at the site of infection. In addition, both cytokines are involved in NLRP3 inflammasome pathways. While IL-18 release is mediated by the NLRP3 inflammasome activation, which recruits caspases-1 to cleave IL-18 pro-peptide to active IL-18 71,72 , GRO-α has been shown to promote the activation of NLRP3 inflammasome 73 . Although dysregulation of these two cytokines were unknown in bvFTD, an increase in IL-18 74,75 and GRO-α 76 have been reported in other neurodegenerative diseases 77,78 . www.nature.com/scientificreports/ Apart from IL-18 and GRO-α, the second components of variance for brain cytokines (Group 2) also contained HGF, M-CSF, MIF, FGF basic, IL-16 and SCGF-β. Of note, GRO-α, HGF, IL-16 and IL-18 levels were all altered (Fig. 3), which is suggestive of their significant physiological roles in bvFTD brain. Apart from SCGFβ-a recently discovered protein for which very little is known-the other cytokines in the clusters are involved in the NLRP3 inflammasome pathway. FGF has been shown to upregulate the NLRP3 inflammasome 79 , while MIF is required for NLRP3 activation 80 . Interestingly, HGF is known to inhibit the NFκB pathway 81 leading to non-expression of RANTES, MCP-1, IL-1β, TNF-α, IL-1 and IL-6 81 . Significantly, the NFκB pathway is known to activate the NLRP3 inflammasome 82 . On the other hand, M-CSF have been shown to activate NFκB 83 . Thus, the cytokines clustered in group 2 of brain cytokines are involved in the NLRP3 inflammasome pathway, either directly or through the NFκB pathway, thus underscoring the importance of NLRP3 inflammasome in the etiology of bvFTD.
The significance of the NFκB pathway in the pathogenesis of FTD was further confirmed by results emerged from the principal component analyses, in which cytokines from the first component of variance for brain cytokines, group 1 (Table 4) www.nature.com/scientificreports/ also have a direct link to the NLRP3 inflammasome: the secretion of IL-1α and β are mediated by the NLRP3 inflammasome 54 , while IL-4 is reported to inhibit inflammasome assembly 118 and SDF-1α inhibits inflammasome activation 119,120 . Notably, cytokines from the entire first components of variance for serum (Group 1), β-NGF, TNFα, G-CSF, IL-1α, IL-2, IL-4, IL-8, IL-3, MIP-1β, IL-9, IL-17A, and IL-7, are all present in the first component of variance for brain cytokines, again confirming the prominence of the NFκB pathway in the etiology of bvFTD. The second component for serum cytokines only consisted of one cytokine, RANTES, and it is also regulated by NFκB 121 . Of note, some of the above cytokines are also responsible for regulation of other cytokines. For example, IL-15 induces IL-8 production 100 , SDF1-α upregulates IL-6 122 and GM-CSF signaling increases IL-1 production 123 , implying secondary regulatory mechanisms within these cytokines. Thus, the current study showed that significantly altered cytokines in bvFTD are all part of an intricate network that revolves around the NLRP3 inflammasome, either directly or via the NFκB pathway. Activation of the NLRP3 inflammasome has been associated with neurodegenerative diseases [124][125][126] , including FTD 55 . Indeed, amyloid-β and α-synuclein were reported to induce NLRP3 inflammasome activation in Alzheimer's 58,59,127 and Parkinson's 60 disease, respectively. In addition, mutant SOD1 and TDP-43 proteins have also been reported to activate NLRP3 inflammasome 128 while aggregated tau 56 and TDP-43 57,129 are also known to activate NLRP3 inflammasome. Interestingly, a recent study implicated NLRP3 inflammasome activation in driving tau pathology 55 . Indeed, inflammasome inhibitors have been shown to inhibit α-synuclein pathology 130 and reduce amyloid-β accumulation 131 in mouse models. Unsurprisingly, there is increasing interest in using NLRP3 inflammasome inhibitors as a therapeutic target for neurodegenerative diseases [132][133][134] . Pilot studies using inhibitors of NLRP3 in mouse models of neurodegenerative diseases have proved this approach effective 130,131,[135][136][137][138] . In an FTD mouse model, the inflammasome inhibitor MCC950 improves inflammation and endoplasmic reticular stress signaling, in addition to partially normalizing the levels of phosphorylated tau 139 .
Taken together, the current study has revealed evidence of cytokine dysregulation in bvFTD serum and brain. In particular, the levels of IL-18 and GRO-α appear to be changed in both serum and brain of bvFTD patients, making these two cytokines possible inflammation biomarkers for bvFTD. Interestingly, these two cytokines are both involved in the NLRP3 inflammasome pathway, which has been associated with other neurodegenerative diseases. Furthermore, principal component analysis performed on serum and brain cytokines have revealed that all significantly altered cytokines are associated with the NLRP3 inflammasome and/or the NFκB pathway, which is a known activator for the NLRP3 inflammasome. Thus, our data show that NLRP3 inflammasome signaling occurs early in the pathogenesis of bvFTD. Given the recent interest in using NLRP3 inflammasome inhibitors as therapeutics against neurodegenerative diseases, and the promising outcomes of these molecules in mouse models, a better understanding on the role of cytokines in NLRP3 inflammasome activation could provide valuable insights into the pathogenesis of bvFTD and its diagnosis and treatment. www.nature.com/scientificreports/

Conclusions
In conclusion, our results showed that cytokine dysregulation is evident in bvFTD brain and serum. Importantly, GRO-α and IL-18 appear to be increased in both serum and brain in bvFTD, making them possible candidates as neuroinflammation biomarkers for bvFTD. The cytokines that are altered in bvFTD serum and/or brain are all related to NLRP3 inflammasome activation or NFκB pathway, which regulates NLRP3. These results therefore suggest that the NLRP3 inflammasome could be important in bvFTD pathogenesis.